Abstract Phosphoinositides (or phosphatidylinositol phosphates, PIPs), resulting from the reversible phosphorylation at the 3, 4 or 5 positions on the inositol ring of Inositol phospholipids, play an important role in various cellular processes, ranging from extracellular signal transduction, vesicle trafficking and cytoskeleton regulation. It is therefore crucial to keep the level of PIPs within the physiological range. Mutations of PIP kinases and phosphatases have been shown to cause various human diseases such as cancers and neurological disorders including Lowe syndrome, Charcot-Marie-Tooth disease, and Alzheimer?s disease. For instance, subtle changes of PTEN level, a 3?-phosphatase of PI(3,4,5)P3, can increase tumorigenicity tremendously. Voltage- sensitive phosphatase (VSP) is a one of the most intriguing players in PIP signaling. VSPs contain a phosphatase domain (PD) that shares sequence homology with PTEN and a membrane spanning voltage- sensing domain (VSD), which controls the PD. Through the lab?s structural analysis of the inter-domain linker and the PD complemented by Voltage Clamp Fluorometry (VCF) analysis of the conformational changes in the VSD triggered by membrane depolarization, a working model has been proposed in which the conformational change of inter-domain linker induced by voltage-driven rearrangement of the VSD displaces a ?gating loop? in the PD to open access to the catalytic site for PIPs at positive voltage. More recent work from the lab suggested that a further gating loop rearrangement or a change in the shape of the catalytic site may account for the substrate selectivity. This proposal seeks out to answer this question by mapping the movement of the intracellular linker domain and the PD, probing the conformational change in the catalytic site and monitoring molecular dynamic at the single molecule level in response to the voltage changes. These together will help visualize the sequential conformational changes during the enzymatic cycle controlled by movements of the voltage sensor. The major approach includes site-specific labeling with fluorescent unnatural amino acid (fUAA) and Cy-dye incorporated non-canonical amino acid in combination with the voltage-clamp fluorometry (VCF) and single-molecule imaging/FRET approaches. Understanding the voltage-gating mechanism of the phosphatase activity in VSPs opens up the possibility of modulating the PIPs level in the cells. This would potentially benefit the treatments for many human diseases related to the malfunction of PIP binding proteins in the cell.